聚腺苷二磷酸核糖聚合酶1在苯并（a）芘诱导人支气管上皮细胞DNA甲基化改变中的作用

Role of poly （ADP-ribose） polymerase 1 on DNA methylation variation induced by B （a）P in human bronchial epithelial cell

目的 观察苯并（a）芘[benzo（a）pyrene,B（a）P]诱导体外细胞DNA甲基化水平改变,探讨聚腺苷二磷酸核糖聚合酶1[poly（ADP-ribose）polymerase 1,PARP1]在该过程中的作用.方法 以1.0、2.0、5.0、10.0、15.0、30.0 μmol/L浓度B（a）P分别处理人支气管上皮细胞（16HBE）及其PARP1缺陷细胞（16HBE-shPARP1）72 h.采用免疫荧光和高效毛细管电泳检测其基因组DNA整体甲基化水平改变,同时动态监测PARP1和DNA甲基转移酶1（DNA methyltransferases 1,DNMT1）表达的变化.结果 16HBE和16HBE-shPARP1细胞基因组整体甲基化百分比（mCpG%）分别为（4.04±0.08）%和（9.69±0.50）%.经5-氮杂脱氧胞苷（DAC）处理72 h后,mCpG%值分别下降为（3.15±0.14）%、（6.07±0.54）%.经B（a）P染毒72 h后,16HBE细胞基因组mCpG%值[B（a）P浓度由低到高]分别为（5.10±0.13）、（4.25±0.10）、（3.91±0.10）、（4.23±0.27）、（3.70±0.15）、（3.08±0.07）;16HBE-shPARP1细胞基因组mCpG%值（浓度由低到高）分别为（10.63±0.60）、（13.08±0.68）、（9.75±0.55）、（7.32±0.67）、（6.90±0.49）、（6.27±0.21）.两种细胞不同处理组间mCpG%差异有统计学意义（F值分别为61.67、60.91,P值均＜0.01）.16HBE细胞各剂量组[B（a）P浓度由低到高]PARP1基因mRNA相对表达水平分别为对照组的141.0%、158.0%、167.0%、239.0%、149.0%、82.9%,差异均有统计学意义（t值分别为11.45、17.32、32.24、33.44、20.21、9.87,P值均＜0.01）;16HBE-shPARP1细胞各剂量组（浓度由低到高）PARP1基因mRNA相对表达水平分别为对照组的169.0%、217.0%、259.0%、323.0%、321.0%、256.0%,差异有统计学意义（t值分别为9.06、15.92、22.68、26.23、37.19、21.15,P值均＜0.01）.当B（a）P染毒剂量达5.0 μmol/L后,16HBE细胞各剂量组（浓度由低到高）DNMT1基因mRNA相对表达水平分别为对照组的125.0%、162.0%、275.0%、233.0%,差异有统计学意义（t值分别为12.74、24.92、55.11、59.07,P值均＜0.01）;当B（a）P染毒剂量达2.0 μmol/L后,16HBE-shPARP1细胞各剂量组（浓度由低到高）DNMT1基因mRNA相对表达水平分别为对照组的135.0%、151.0%、180.0%、229.0%、186.0%,差异有统计学意义（t值分别为23.82、40.17、32.69、74.85、46.76,P值均＜0.01）.结论 B（a）P诱导的16HBE细胞基因组整体甲基化水平降低可能是其恶性转化过程中早期重要的分子事件,PARP1可通过抑制DNMT1的酶活性来调节B（a）P诱导的16HBE细胞DNA甲基化水平,这种效应可因PARP1的缺失而缓解.

Objective To investigate DNA methylation variation in human cells induces by B （a）P, and to explore the role of PARP1 during this process. Methods The changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B （ a ） P （ 1.0, 2. 0, 5.0, 10. 0, 15.0, 30. 0 μ mol/L ） were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP1 and DNMT1 were monitored dynamically. Results The percentage of methylated DNA of overall genome （ mCpG% ） in 16HBE and 16HBE-shPARP1 cells were separately （4. 04 ±0. 08） %and （9. 69 ±0. 50）%. After being treated by 5-DAC for 72 hours,mCpG% decreased to （3.15 ±0. 14）%and （6. 07 ± 0. 54 ） %. After both being exposed to B （a） P for 72 hours, the mCpG% in 16HBE group （ ascending rank ） were separately （ 5. 10 ± 0. 13 ）, （ 4. 25 ± 0. 10 ）, （ 3.91 ± 0. 10 ）, （ 4. 23 ± 0. 27 ）,（3.70 ± 0. 15 ）, （ 3.08 ± 0. 07 ）; while the figures in 16HBE-shPARP1 group （ ascending rank ） were respectively （10.63 ±0.60）, （13.08 ±0.68）, （9.75 ±0.55）, （7.32 ±0.67）, （6.90 ±0.49） and （6. 27 ±0. 21 ）. The difference of the results was statistically significant （ F values were 61.67 and 60. 91,P〈 0.01 ） . For 16HBE group, expression of PARP1 and DNMT1 were 141.0%, 158.0%, 167.0%,239. 0%, 149. 0% ,82. 9% and 108. 0%, 117.0%, 125.0%, 162. 0% ,275. 0% ,233.0% comparing with the control group, whose difference also has statitical significance （t values were 11.45,17. 32,32. 24,33.44,20.21 and 9. 87 ,P 〈 0. 01 ）. For 16HBE-shPARP1 group, expression of PARP1 and DNMT1 were 169.0% ,217.0%, 259.0%, 323.0% , 321.0% , 256.0% and 86.0% , 135.0% , 151.0% , 180.0%,229. 0%, 186. 0% comparing with the control group,with statitical significance （t values were 9. 06,15. 92,22. 68,26. 23,37. 19 and 21.15, P 〈 0. 01 ）. When the dose of B （a） P reached 5.0 μmol/L, the mRNA expression of DNMTI in 16HBE group （ascending rank） were 125.0%, 162. 0% ,275.0% ,233.0% times of it in control group, with statistical significance （ t values were 12. 74,24.92,55. 11,59. 07, P 〈 0. 01 ）;while the dose of B（a） P reached 2.0 μmol/L, the mRNA expression of DNMT1 in 16HBE-shPARP1 group were 135.0%, 151. 0%, 180. 0% ,229.0%, 186. 0% of the results in control group, and the differences were statistically significant （ t values were 23. 82,40. 17,32. 69,74. 85,46. 76, P 〈 0. 01 ）. Conclusion The hypomethylation of 16HBE cells induced by B（a）P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.